Projects

Currently, there are three main areas of research in my lab focusing on intracellular and extracellular regulation of cartilage tissue development and homeostasis.

1. MicroRNAs regulating cartilage development and homeostasis

Research on small non-coding microRNAs (miRNAs) has accelerated over the past decade. These molecules are now known to be important regulators of many cellular processes through their actions of gene suppression, thereby potentially affecting entire cellular pathways. They are also known to be dysregulated in a number of pathological conditions and are being investigated as potential therapeutic targets to treat certain diseases.

With respect to cartilage, we recognized that there was a lack of detailed information on in vivo expression of miRNAs as well as information on miRNAs that may regulate specific phases of chondrocyte differentiation. Toward this end, we utilized human embryonic limb tissue sections at a stage of development prior to endochondral bone formation and carried out laser capture microdissection to isolate regions containing progenitor chondrocytes (PC), differentiated chondrocytes (DC) and hypertrophic chondrocytes (HYP) (see Figure below). We then carried out TaqMan®-based OpenArrays® to determine both highly expressed and differentially-expressed miRNAs between chondrocytes at distinct stages of differentiation. This work has been published in PLoS ONE, 2013. Thus, we have generated an important human miRNA database containing unique expression information with respect to cartilage development.

We are now continuing this line of research by selecting candidate miRNAs to determine function in regulating MSC chondrogenic differentiation. We are also gearing up to utilize induced pluripotent stem cells (iPSCs) as a means to generate MSCs for our assays. Future studies will involve utilizing OA patient-derived iPSCs. We are also interested in generating miRNA over-expression libraries to determine strategies to induce differentiation of MSCs (or even fibroblasts) toward the chondrocyte lineage without the growth factor requirement.

In addition to miRNAs regulating development/differentiation, we are also pursuing studies to determine their roles in regulating homeostasis in mature cartilage tissue. We are developing murine joint loading models of post-traumatic osteoarthritis (PTOA) to find miRNAs that are responsive to supraphysiologic loading and/or inflammation induced by specific loading regimes. The overall goals of this work are to: i) determine a miRNA expression signature in cartilage associated with joint trauma, ii) identify miRNAs that may function to regulate anabolic or catabolic processes in chondrocytes and iii) modulate expression of specific miRNA(s) in vivo to attempt to slow down or stop cartilage degradation or even induce cartilage repair/regeneration.

Type II collagen is the major collagen in the extracellular matrix (ECM) of articular cartilage and provides this tissue with important tensile properties. This fibrillar collagen is synthesized as a procollagen and, through the process of pre-mRNA alternative splicing, different isoforms exist depending on the differentiation status of the chondrocyte. Progenitor chondrocytes synthesize “embryonic” type II collagen isoforms containing a conserved exon 2-encoded cysteine-rich domain, while differentiated chondrocytes produce type II procollagens devoid of exon 2.

Utilizing a splice site targeting approach, we have generated knock-in transgenic mice that exclusively express embryonic (IIA) isoforms of type II procollagen, even in mature cartilage tissue where it is not normally expressed (see Figure below). Surprisingly, heterozygous and homozygous knock-in mice do not display any overt skeletal abnormalities. However, further characterization has shown persistence of IIA collagen isoforms in the ECM of mature articular and growth plate cartilage (Matrix Biology, 2012).

Although the transgenic knock-in mice appear skeletally normal, we have found differences in collagen ultrastructure at the electron microscopy level (unpublished observations). Whether this translates to a biomechanically inferior (or superior) tissue has yet to be determined. Current studies involve analysis of articular cartilage from aged (1 yr old) knock-in mice and induction of OA in 10wk old transgenic mice by destabilization of the medial meniscus (DMM). Here we will address the compelling question of whether or not an ECM derived from embryonic collagens could be resistant / less prone to cartilage breakdown.

Many efforts are being put into tissue engineering approaches to attempt to repair or regenerate articular cartilage. These efforts include generation of scaffolds in combination with stem cells / chondrocytes and growth factors. We recognize that the basic scientist-bioengineer collaboration is critical to better understand how scaffold biomaterials may affect the response of mesenchymal stem cells (MSCs) to growth factors, or other differentiation-inducing factors. In collaboration with Don Elbert, we addressed the simple question of how adult bone marrow-derived MSCs (BMSCs) respond to TGF-β3-induced chondrogenesis in the presence of poly(ethylene glycol) (PEG) microspheres. We found dramatic differences in ECM production by MSCs cultured in the presence of microspheres compared to MSCs cultured in condensations without microspheres. For example, there was more widespread production of proteoglycans (detected by Safranin-O staining) in the MSC cultures containing microspheres (see Figure below). We also detected changes in the production and localization of type I, II and X collagens as a result of microsphere co-culture (see our Biomaterials, 2011 paper). We are interested in continuing these studies to determine why the biomaterial microenvironment apparently has a profound effect on MSC differentiation with respect to the type of ECM being generated.